The Journal of Neuroscience, November 10, 2004 • 24(45):10103–10110 • 10103

Behavioral/Systems/Cognitive Role of Neurokinin 3 Receptors in Supraoptic and Neurons

Heather E. Howe, Suwit J. Somponpun, and Celia D. Sladek Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado 80262

Neurokinin 3 receptors (NK3-Rs) are expressed in the supraoptic nucleus (SON), and SON is innervated by (SP)-expressing A1 neurons in the medulla. Because SP stimulates vasopressin (VP) and oxytocin release from explants of the hypothalamo- neurohypophyseal system (HNS), two hypotheses were tested: (1) SP-stimulated VP release is mediated by NK3-Rs, and (2) stimulation of the A1 pathway by hypotension activates SON NK3-Rs. Senktide, an NK3-R agonist, stimulated VP release from HNS explants, but neither a neurokinin 1 antagonist [L732,138 (N-acetyl-L-tryptophan 3,5-bis(tri-fluoromethyl)benzyl ester)] nor two NK3-R antagonists (SB222200 and SB235375) prevented SP-stimulated VP release. Because the affinity of these antagonists for rat NK-Rs may limit their efficacy, NK3-R internalization was used to assess the ability of SP to activate SON NK3-Rs. Senktide, SP, or vehicle was microinjected above SON. The brain was perfused 5 min after injection and stained for NK3-R immunoreactivity. Using confocal micros- copy, the number of NK3-R-immunoreactive (-IR) endosomes was counted in a 5.6 2 ␮ region of cytoplasm in SON neurons. Senktide, but not SP or vehicle, significantly increased the number of NK3-R-IR endosomes in the cytoplasm. When hypotension was induced with hydralazine, NK3-R internalization was observed within 5 min ( p Ͻ 0.005). A decrease in cytoplasmic NK3-R immunoreactivity was observed within 15 min of hypotension. Unexpectedly, both senktide and hypotension resulted in translocation of NK3-R-IR immuno- reactivity to the nucleus. Thus, although these studies do not identify SP as the NK3-R ligand, they do provide evidence for hypotension- induced release of an endogenous tachykinin in SON and evidence suggesting a role for NK3-Rs in transcription regulation. Key words: oxytocin; receptor; substance P; supraoptic; vasopressin; neurokinin 3

Introduction for these receptors and their importance to physiological stimu- The tachykinin family includes substance P (SP), neuro- lation of VP and OT release from the neurohypophysis has not A, and (NKB). These interact with been determined. Immunocytochemical studies reported a few three neurokinin receptors (NK1-R, NK2-R, and NK3-R). Sub- NKB-immunoreactive (-IR) fibers in SON (Lucas et al., 1992), stance P, , and NKB exhibit the highest affinity for and one study reported NKB-positive cells in SON (Hatae et al., NK1-R, NK2-R, and NK3-R, respectively, but each ligand can 2001); however, although immunoreactivity to a 30-residue pep- activate the other receptor types if present at a sufficiently high tide of the NKB protein precursor was reported in SON (Mark- concentration. These receptors are G-protein-coupled receptors steiner et al., 1992; Hatae et al., 2001), NKB-IR cell bodies and (GPCRs), all of which can activate both phosphotidylinositol hy- mRNA were not detected in SON with in situ hybridization drolysis and the cAMP signal cascade (Nakajima et al., 1992). (Warden and Young, 1988; Lucas et al., 1992; Marksteiner et al., NK3-Rs are highly expressed in the supraoptic nucleus (SON) 1992; Merchenthaler et al., 1992). The presence of NKB- and the paraventricular nucleus (PVN) (Ding et al., 1996, 1999; expressing cells adjacent to SON (Marksteiner et al., 1992) may Shughrue et al., 1996; Mileusnic et al., 1999). Activation of these account for NKB mRNA detected by Western blot (Hatae et al., receptors by intracerebroventricular injection of NKB or a selec- 2001). tive NK3-R agonist, senktide, results in Fos expression in SON In contrast, SON and PVN are innervated by SP fibers (Hol- and PVN (Ding et al., 2000; Smith and Flynn, 2000; Spitznagel et zbauer et al., 1984; Sladek and Armstrong, 1985; Bittencourt et al., 2001), as well as antidiuresis (Eguchi et al., 1996) and periph- al., 1991). These fibers originate principally from the A1 catechol- eral vasopressin (VP) and oxytocin (OT) release (Polidori et al., amine neurons in the ventral medulla, and 15–25% of these neu- 1989; Bealer and Flynn, 2003); however, the endogenous ligand rons express SP (Bittencourt et al., 1991). This pathway transmits information about decreases in blood volume and pressure Received Aug. 2, 2004; revised Sept. 15, 2004; accepted Sept. 23, 2004. (Cunningham and Sawchenko, 1991). The report that SP is re- ThisworkwassupportedbyNationalInstitutesofHealthGrantR01-NS44835toC.D.S.Thetechnicalassistanceof SteveFadulintheUniversityofColoradoHealthScienceCenterLightMicroscopyFacilityisgratefullyacknowledged. duced in SON after hemorrhage (Feuerstein et al., 1984) supports We thank Drs. David Brooks and Douglas W. Hay of GlaxoSmithKline for their help in obtaining the NK3-receptor release of SP in response to activation of the A1 pathway by hy- antagonists and their advice on the use of the antagonists. potension. SP is a potent stimulus for VP and OT release with 10 Correspondence should be addressed to Dr. Celia D. Sladek, Department of Physiology, University of Colorado ␮M SP inducing a large and sustained increase in VP and OT Health Science Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.3164-04.2004 release from explants of the hypothalamo-neurohypophyseal sys- Copyright © 2004 Society for Neuroscience 0270-6474/04/2410103-08$15.00/0 tem (Kapoor and Sladek, 2001; Juszczak and Stempniak, 2003). 10104 • J. Neurosci., November 10, 2004 • 24(45):10103–10110 Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons

Because the magnocellular neurons express NK3-Rs but not NK1-Rs (Nakaya et al., 1994), and because SP is an agonist for NK3-Rs, SP may be the endogenous ligand for the NK3-Rs in SON. NK3-R antagonists were used to test the hypothesis that NK3-Rs mediate SP stimulation of VP release from hypothalamo- neurohypophyseal system (HNS) explants. NK3-R internalization was used to test the hypothesis that a tachykinin is released when the A1 catecholamine pathway is activated by hypotension. Materials and Methods Animals Male Sprague Dawley rats [Crl:CD(SD)Br (Charles Rivers Laboratories, Wilmington, MA)] were maintained on a 12 hr light/dark cycle with lights on at 6 A.M. and ambient temperature at 21 Ϯ 1°C. Standard rat Figure 1. Demonstration of the technique used to quantitate NK3-R internalization. The chow and water were available ad libitum throughout the experiment. All originalimage(A)wasconvertedtograyscale(B),andauniformthresholdwasapplied(red)to protocols used were performed in accordance with the National Institutes determinetheimmunoreactiveregionstobecounted.Theimmunoreactivespotswithina5.6 2 of Health Guidelines for the Care and Use of Laboratory Animals and were ␮m region (white box) were counted. In this example, 13 endosomes were counted. Scale approved by the Institutional Animal Care and Use Committee of the bar, 10 ␮m. University of Colorado Health Science Center. collected from each explant. The standards and samples were incubated HNS explant perifusion experiments 125 Male rats (125–149 gm) were decapitated and explants of the HNS were for 72 hr at 4°C in the presence of 5000 counts per minute of I- prepared as described previously (Sladek and Knigge, 1977). The brain arginine vasopressin (NEN, Boston, MA). Antibody-bound VP was sep- and pituitary were removed from the skull using a caudal approach to arated from free hormone with dextran-coated charcoal, and the amount 125 ensure that the pituitary stalk was intact. The anterior pituitary was re- of I-VP in the pellet was determined with a gamma counter. The moved under a dissecting microscope. After the meninges (dura mater picograms per milliliter were obtained by comparing samples with a and arachnoid) was gently removed, a triangular block of tissue was standard curve of known concentrations of VP. All samples from a given removed from the ventral hypothalamus by cutting rostral to the optic experiment were assayed at the same time. The minimum sensitivity was chiasm, lateral to either side of the median eminence, and undercutting 1.0 pg per tube for VP. at a depth of 1–2 mm. The explants included the magnocellular neurons Statistical analysis. As mentioned previously, each explant was allowed of the SON with their axonal projections extending through the median to equilibrate for 4 hr before exposure to drugs. Basal hormone release eminence and terminating in the neurohypophysis. Using a dissecting was calculated for each explant as the mean hormone release at the end of microscope, the explants were examined to ensure that the neurohy- this equilibration period. Hormone content is expressed as a percentage Ϯ pophyseal stalk was intact. Also included in the explant are the suprachi- of this basal value. Results are expressed as mean SEM. Statistical asmatic, arcuate, and ventral portions of the ventromedial, preoptic, and significance was determined on log10-transformed data by ANOVA with periventricular nuclei as well as the organum vasculosum of the lamina repeated measures followed by simple main effect analysis to establish terminalis. specific group differences at individual time points. Level of significance Ͻ Each explant was placed in a 500 ␮l perifusion chamber, maintained at was set to p 0.05. 37°C in the multiple microchamber unit (Cellex Biosciences, Minneap- NK3 receptor internalization olis, MN), and perifused with F12 nutrient mixture (Sigma, St. Louis, Surgical procedures. Male rats (275–300 gm) were anesthetized intraperi- MO) fortified with 20% fetal calf serum, 1 mg/ml glucose, 50 ␮U/ml 4 toneally with avertin (0.4 mg of 2,2,2 tribromoethanol/gm body weight, penicillin, 50 ␮g/ml streptomycin, and 1 ϫ 10 Ϫ M bacitracin. Bacitra- prepared in saline; Sigma Aldrich). Venous and arterial femoral catheters cin was added to the medium to prevent hormone degradation (Sladek were implanted and exteriorized at the base of the neck. The catheters and Armstrong, 1987). The final osmolality of the culture medium was

295–300 mOsm/kg H2O. The medium was warmed (37°C) and gassed (95% O2,5%CO2) immediately before being placed in the explant cham- ber. Six explants were perifused simultaneously at a rate of ϳ2.0 ml/hr, and outflow from the chambers was collected individually in 20 min intervals using a six-place fraction collector kept in a refrigerator (4°C) for subsequent measurement of VP or OT concentration. Radioimmu- noassay (RIA) was used to determine VP concentration in these samples. Hormone release was allowed to stabilize for 4 hr before exposure to any experimental conditions. Subsequently, explants were perifused with basal medium or the indicated concentrations of SP (AnaSpec, San Jose, CA) or senktide (Tocris Cookson, Ellisville, MO). Explants were exposed to an NK1 antagonist, N-acetyl-L-tryptophan 3,5-bis(tri-fluoromethyl) benzyl ester (L732,138) (Sigma) (Cascieri et al., 1994) or one of two NK3-R antagonists, SB222200 and SB235375 (Sarau et al., 2000; Hay et al., 2002) (generously provided by GlaxoSmithKline, Research Triangle Park, NC), 30 min before the addition of SP. Drugs and peptides were dissolved directly into the medium with the exception of SB222200, which was dissolved in dimethylsulfoxide (DMSO). Control explants were exposed to the same concentrations of DMSO (n ϭ 3–6 per group). Radioimmunoassay. VP concentration in the perifusate was deter- Figure 2. Effect of an NK3-R agonist, senktide, on VP release from HNS explants. After a 4 hr mined by RIA as described previously (Kapoor and Sladek, 2001). The equilibration period, senktide (0.1 ␮M) was added to the perifusate of one group of HNS ex- antisera was generated in conjunction with Arnel Products (Brooklyn, plants. Perifusate was collected at 20 min intervals for determination of VP content by RIA. VP NY) and used at a final dilution of 1:100,000. The buffer was 0.1 M PBS, content was increased significantly in the perifusate from the senktide-exposed explants com- pH 7.6, with 1 mg/ml bovine serum albumin and 1 mg/ml sodium azide. pared with the controls (*p Ͻ 0.05) for the duration of the exposure. Basal release for control The assay was performed on 100 and 50 ␮l aliquots of each fraction group was 156 Ϯ 61 pg/ml (n ϭ 4); for the senktide group, it was 158 Ϯ 45 pg/ml (n ϭ 5). Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons J. Neurosci., November 10, 2004 • 24(45):10103–10110 • 10105

were composed of a 6 inch piece of PE50 polyethylene tubing melted to either a 40 mm (arterial) or a 30 mm (venous) piece of PE10 tubing. Arterial catheters were filled with 200 U/ml heparanized saline to main- tain patency of the line. In some animals, a cannula (23 gauge) was implanted unilaterally above the SON using stereotaxic coordinates (1.0 mm caudal, 1.4 mm lateral, 9.0 mm ventral relative to bregma). The cannula was held in place with dental acrylic and two small screws. Sur- geries were performed 3 d before the experiment. Blood pressure measurements. On the morning of the experiment, the arterial catheter was connected to a pressure transducer, and arterial pressure was monitored for 15 min. The animal was then anesthetized Figure3. EffectofNK1-Rantagonist,L732,138,onSP-stimulatedVPrelease.Theantagonist with urethane [1.25 gm urethane/kg body weight (Sigma Aldrich); deliv- did not significantly alter the VP response to either 10 ␮M (A)or5␮M (B) SP. Basal release for ered via the arterial catheter], and arterial pressure was monitored for 1 these groups of explants was as follows: A,46Ϯ 9 pg/ml for SP alone and 45 Ϯ 12 pg/ml for additional hour before the following protocols. SPϩL732,138; B,88Ϯ 12 pg/ml for SP alone and 99 Ϯ 18 pg/ml for SPϩL732,138. n ϭ 5or SP–senktide protocol. One hour after urethane was administered, a 0.2 6 per group. ␮l microinjection of substance P [100 ␮M in PBS (Tocris Cookson); n ϭ 10], senktide [79 ␮M in PBS (Tocris Cookson); n ϭ 9], or PBS (n ϭ 12) was administered through the brain cannula using a 5 ␮l Hamilton sy- ringe. Five minutes after the injection, rats were perfused transcardially sections through SON that were analyzed on the injected side were within ␮ with 80 ml of 0.1 M PBS followed by 150 ml of 4% paraformaldehyde 400 m of the tip of the injection cannulas (see Fig. 5E for an example of (PFA) in 0.1 M phosphate buffer. After perfusion, the brain was removed a cannula placement immediately above SON). This was not significantly and placed in 30% sucrose in 4% PFA for 3 hr and then in 30% sucrose in different among the PBS, SP, and senktide groups. In the hypotension PBS for at least 3 d. experiment, 30 cells were analyzed in three sections through SON in each 2 Hypotension experiment. After blood pressure was allowed to stabilize animal. We chose to sample a consistent area (5.6 ␮m) within each cell for 1 hr under urethane anesthesia, hydralazine (10 mg/kg; Sigma Al- to eliminate the differences in cytoplasmic area between cells. Cells were drich) or saline was administered via the venous catheter. At 5, 15, or 60 selected randomly from the visual field (see Fig. 5D,E), but they had to min after this treatment, rats were perfused transcardially with 80 ml of meet the following criteria. The entire cell had to be present in the section 0.1 M PBS followed by 150 ml of 4% PFA in 0.1 M phosphate buffer. After as determined from the examination of the z-stack, and the z-section perfusion, the brain was removed and placed in 30% sucrose in 4% PFA analyzed had to include the cell nucleus and a sufficient area of cytoplasm 2 for 3 hr and then in 30% sucrose in PBS for at least3d(n ϭ 5 per group to accommodate the 5.6 mm counting square shown in Figure 1. except the 5 min PBS group, which was n ϭ 4). Statistical analysis. Student’s paired t test was used to compare endo- Histology. Brains were sectioned at 30 ␮m with a cryostat and stored in some counts between the injected and noninjected SONs. Nonpaired t a cryoprotectant solution (30% sucrose, 30% ethylene glycol, 1% poly- tests were used to compare internalization between the saline- and vinylpyrrolidone in 0.1 M phosphate buffer) at Ϫ20°C until they were hydralazine-treated rats. One-way ANOVA was used to assess the time processed for staining. All rinses and incubations except the antibody course of the internalization response to hydralazine-induced incubation were performed at room temperature. On the day of staining, hypotension. all sections were rinsed in 0.1 M PBS with 0.2% Triton-X (PBS/TriX) for 1 hr. They were then incubated in 0.5% sodium borohydride prepared in Results 0.1 M PBS for 20 min followed by 1 hr of rinsing in PBS/TriX. Next, the HNS explant perifusion experiments sections were incubated in 0.3% H O prepared in water followed by 30 2 2 Effect of NK3-R agonist min of rinsing in PBS/TriX. The sections were then placed in 1.5% goat Senktide was used as a selective NK3-R agonist to evaluate the serum in PBS/TriX for 1 hr. They were then incubated in a rabbit poly- effect of NK3-R activation on VP release from HNS explants. As clonal antibody to the NK3-R [NB300–102 (Novus Biologicals, Littleton, ␮ CO); 1:10,000 dilution] for 72 hr at 4°C. The sections were then rinsed for shown in Figure 2, senktide (0.1 M) significantly increased VP 30 min and incubated in goat anti-rabbit IgG antibody labeled with Alexa release compared with control explants perifused with basal me- Fluor 488 (Molecular Probes, Eugene, OR; 1:400 dilution in PBS/TriX) dium (F ϭ 18.82; p Ͻ 0.005). The response was sustained for the for 1 hr. After a 30 min rinse in PBS/TriX, the sections were placed in 300 duration of exposure (ϳ1.5 hr). Higher concentrations of senk- nM 4Ј,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular tide did not result in a greater increases in VP release (data not Probes) for 5 min. The sections were then rinsed in 0.1 M PBS, mounted shown). on poly--treated slides, and coverslipped with ProLong Antifade (Molecular Probes). The NK3-R antibody was raised against a synthetic peptide from the C terminus of the rat NK3-R (aa 438–452). Staining was not observed when the primary antibody was omitted. Image collection. Confocal images were acquired using a Zeiss LSM 510 NLO laser scanning confocal microscope with argon (488 nm) and tita- nium:sapphire (795 nm) lasers, using a 63ϫ oil objective. For each image, Z stacks of 12 images were obtained with each image taken 2.4 Ϯ 0.1 m apart. Quantification of NK3-IR internalization. An investigator, blind to the animal’s blood pressure status or drug treatment, quantified internaliza- tion using NIH ImageJ software. First, each image was converted to gray scale. As demonstrated in Figure 1, after application of a uniform thresh- old, the number of endosomes in a 5.6 2 mm region within the cytoplasm Figure 4. Effect of NK3-R antagonists, SB235375 (A) and SB222200 (B), on SP-stimulated was counted. In the SP–senktide injection experiments, 10 cells were VP release. The antagonists (500 ␮M) were added to the perifusate 30 min before SP (5 ␮M). analyzed in two sections through each SON. Thus 40 cells were analyzed Neither antagonist prevented SP-stimulated VP release. Although there was a trend for both in each rat (20 from the injected side and 20 from the noninjected side). antagonists to reduce basal VP release before the addition of SP, only one time point met The locations of these cells spanned the dorsal–ventral and medial–lat- significance with independent Student’s t test (*p Ͻ 0.02; **p ϭ 0.054). Basal release was as eral extent of the nucleus (see Fig. 5D,E, for examples of visual field); follows: A, 48.6 Ϯ 16.1 pg/ml for SP alone, 68.8 Ϯ 33 pg/ml for SPϩSB235375; B,25Ϯ 5.5 therefore, both VP and OT neurons were included in the analysis. The pg/ml for SP alone, 29.4 Ϯ 6.3 pg/ml for SPϩSB222200. 10106 • J. Neurosci., November 10, 2004 • 24(45):10103–10110 Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons

Figure5. ImmunocytochemicallocalizationofNK3immunoreactivity(green)inSONofratsinjectedwithPBS,senktide,orSPimmediatelyabovetheSONononesideofthebrainisshowninA–C. In the PBS-injected rats, there is prominent localization of the NK3 immunoreactivity to the in both the injected (A2) and noninjected (A1) sides. In the senktide-injected rat, the noninjected side (B1) displays membrane localization, whereas the injected side (B2) displays NK3 immunoreactivity internalized to the cytoplasm. NK3 immunoreactivity is localized to the membrane in both the SP-injected side (C2) and the noninjected side (C1). The cell nuclei were stained with DAPI (blue). White arrows indicate punctate NK3 immunoreactivity localized to the nucleusafterthesenktideinjection(B2).NuclearlocalizationwasnotobservedaftertheSPinjection(C2).DshowstheregionofSONdepictedinB2.Eshowsarepresentativecannula(CAN)placement with the injector (INJ) extending to immediately above SON. The cannula tract is not visible in D but was present in the section rostral to this level. Scale bars: A–C,10␮m; D, E, 100 ␮m.

Effect of NK1-R antagonist Because SP has the highest affinity for NK1-Rs, the ability of an NK1-R antagonist, L732,138, to block SP-stimulated VP release was evaluated. The antagonist was added to the perifusate 30 min before SP. As shown in Figure 3, SP caused a dose-dependent increase in VP release comparable with that observed previously (Kapoor and Sladek, 2001), with 5 ␮M SP resulting in a sustained increase of ϳ250% of basal and 10 ␮M SP causing a 300–400% ϭ increase. These increases are statistically significant (F5 ␮M 10.8, Ͻ ϭ Ͻ p 0.0001; F10 ␮M 10.04, p 0.0001) and were sustained for the duration of exposure to SP. L732,138 (100 ␮M) did not sig- nificantly alter the response to either 5 or 10 ␮M SP, suggesting that the SP effect is not caused by activation of NK1-Rs. Effect of NK3-R antagonists Because NK3-Rs are expressed in SON and because SP can act as a ligand for NK3-Rs, albeit with lower affinity than for NK1-Rs (Nakajima et al., 1992), the ability of NK3-R antagonists to block SP stimulation of VP release was evaluated. Two NK3-R antago- Figure 6. Quantification of the effect of PBS, senktide, and SP injections immediately above nists, SB235375 and SB222200, were used. As shown in Figure 4, SON on the cytoplasmic localization of NK3-IR endosomes. Senktide, but not PBS nor SP, in- creased the number of endosomes on the injected side (*p ϭ 0.015). Data are expressed as the VP response to 5 ␮M SP was not significantly altered by either mean Ϯ SEM number of endosomes in a 5.6 2 ␮m region of cytoplasm per neuron per animal. antagonist. The NK3-R antagonists were added to the perifusate n ϭ 9–12 animals per group. 30 min before SP to assess the effect of the antagonists on basal VP release. Although there was a tendency for VP release to be lower in the NK3-R antagonists groups before the addition of SP, this NK3-R antagonists do not provide evidence that SP-stimulated did not result in a significant difference between the groups or a VP release is mediated by NK3-Rs. significant interaction between time and treatment in the two- way, repeated-measure ANOVA in either experiment, and only NK3 receptor internalization in SON one data point met the criteria for a statistical significant differ- NK1-Rs and NK3-Rs, as well as several other G-protein-coupled ence by independent Student’s t test (4.6 hr in the SB222200) receptors, undergo rapid endocytosis on ligand binding (Ko- (Fig. 4B)(*p Ͻ 0.05). Thus, although VP release can be stimu- bilka, 1992; Colin et al., 2002). Internalization of NK receptors lated by an NK3-R agonist (senktide), the experiments with has been used as an index of neurokinin release in response to Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons J. Neurosci., November 10, 2004 • 24(45):10103–10110 • 10107

of the injection compared with the noninjected side ( p ϭ 0.015), but SP did not increase cytoplasmic localization of NK3 immu- noreactivity. Because cells located throughout the dorsal–ventral extent of SON were analyzed in each section of SON (Fig. 5D,E), it is probable that senktide induced NK3-R internalization in both VP and OT neurons. Effect of hydralazine-induced hypotension on NK3-R localization The effect of urethane anesthesia and saline or hydralazine injec- tions on mean arterial blood pressure is shown in Figure 7. Blood pressure was not altered by the urethane or saline injections but decreased to 49 Ϯ 2 mmHg by 5 min and to 30 Ϯ 2 mmHg by 15 min after hydralazine. It remained low without additional hy- dralazine for 1 hr after the injection. Heart rate was not signifi- cantly altered by the hydralazine injection. This probably reflects inhibition of the baroreflex by urethane (Shimokawa et al., 1998), because in previous studies using conscious rats, heart rate was Figure7. Effectofhydralazineonmeanarterialbloodpressure.Hydralazinerapidlyinduced increased after hydralazine (Stocker et al., 2000). As seen in Fig- hypotension that was sustained for as long as 60 min. Urethane anesthesia did not alter blood ure 8, the NK3 immunoreactivity was primarily localized to the pressure. Animals were anesthetized 1 hr in advance of hydralazine administration so that the brain could be perfused shortly after the induction of hypotension. cell membrane in the SON of saline-injected rats at all time points. There was a significant increase in cytoplasmic localiza- tion of the NK3 immunoreactivity at 5 min after hydralazine physiological and pharmacological stimuli (Colin et al., 2002; (Figs. 8A2, 9) ( p ϭ 0.005). Cytoplasmic localization decreased Sabino et al., 2002). We used NK3-R internalization to assess the rapidly and was not significantly different from that in saline- hypotheses that (1) SP acts as a ligand for NK3-Rs in SON and (2) injected animals at 15 or 60 min of hydralazine-induced hypo- NK3-Rs in SON are activated in response to hypotension. tension. It was significantly reduced compared with the 5 min hypotensive group (Kruskal–Wallis one-way ANOVA on ranks; Effect of local injection of senktide or SP on NK3-R localization H ϭ 14.347; p ϭ 0.014). These data indicate that a ligand for the Local injection of senktide (79 ␮M), but not PBS or SP (100 ␮M), induced an increase in the number of NK3-IR endosomes in the NK3-R is released in response to hydralazine-induced hypoten- cytoplasm in SON neurons at 5 min after injection. As shown in sion. Because hydralazine inhibits production of II Figure 5, NK3 immunoreactivity was localized primarily to the (Stocker et al., 2000), activation of the A1 pathway from the cell membrane of SON neurons on the noninjected side of the caudal ventrolateral medulla to SON is likely to mediate VP and brains. It was also localized to the cell membrane on the side OT release induced by hydralazine (Day and Sibbald, 1989; injected with PBS and SP. Quantification of the number of cyto- Schiltz et al., 1997). plasmic endosomes is shown in Figure 6. Senktide-injected rats Nuclear localization of NK3 immunoreactivity displayed significantly more internalized endosomes on the side After senktide injection above SON and for up to 1 hr after hydralazine-induced hypotension, NK3 immunoreactivity was observed in the nu- clei of SON neurons (Figs. 5B2,8A2–C2). DAPI staining of the nucleus, collection of confocal images through the z-axis of the nucleus, and the presence of NK3 immu- noreactivity at the level of nucleolus pro- vided evidence that these NK3-IR particles were located within the nucleus and not simply in the perinuclear region. Perinu- clear localization of GPCR or incorpora- tion of GPCR into the nuclear envelope is seen as a distinct rim of immunoreactivity around the nucleus in confocal images (Bhattacharya et al., 1999; Gicquiaux et al., 2002). Perinuclear localization of GPCR is a common feature of GPCR sorting after endocytosis (Sorkin and Von Zastrow, 2002), and prostaglandin receptors have been visualized in the nuclear envelope (Bhattacharya et al., 1999). As seen in Fig- ures 5B2 and 8A2–C2, however, NK3 im- Figure 8. Localization of NK3-Rs in SON in response to hypotension at 5, 15, and 60 min. Membrane localization of NK3 munoreactivity did not rim the nucleus, immunoreactivity (green) is evident at all time points in the saline-injected, normotensive animals (A1–C1). Five minutes of and imaging through the z-plane of the hypotension induces NK3-R internalization (A2), which is decreased by 15 min (B2) and remains low after 60 min of hypotension nucleus allowed verification that the im- (C2). The nuclei are stained with DAPI (blue). Note the prominent nuclear localization of NK3 immunoreactivity that is evident at munoreactivity was not above or below 5 min of hypotension and remains evident at 15 and 60 min of hypotension (white arrows). Scale bar, 10 ␮m. the nucleus. Quantitation of NK3-IR nu- 10108 • J. Neurosci., November 10, 2004 • 24(45):10103–10110 Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons

Marvizon et al., 2003). Ligand-induced NK3-R internalization has also been demonstrated (Colin et al., 2002; Schmidlin et al., 2003), but to our knowledge, NK3-R internalization has not been used previously to identify the nature of the involved in specific physiological responses. In the current stud- ies, rapid internalization of the NK3-R occurred in SON after hydralazine-induced hypotension. Hypotension activates SON neurons and is a potent stimulus for VP and OT release from the neural lobe (Stricker and Verbalis, 1986; Smith and Day, 2003). Although the A1 pathway has been identified as the pathway that carries information about decreases in blood pressure from the brainstem to the SON (Day and Sibbald, 1989), the neurotrans- mitter(s) responsible for hypotension-stimulated VP release has Figure 9. Quantification of the time course of NK3-R internalization during hydralazine- not been unequivocally identified. The A1 pathway was originally induced hypotension. The number of cytoplasmic NK3-IR endosomes was significantly in- identified as a catecholaminergic pathway, but noradrenergic an- creased by 5 min of hypotension in hydralazine-treated rats (*p ϭ 0.005; n ϭ 5) compared tagonists do not block activation of SON neurons by hypotension with normotensive rats (n ϭ 4). By 15 min, cytoplasmic NK3 immunoreactivity was signifi- (Day et al., 1990). Furthermore, other and cantlydecreased(nϭ5)andremainedlowafter60minofhypotension(nϭ5;**pϽ0.05by are colocalized in the A1 catecholamine neurons post hoc Newman–Keuls analysis). (Blessing et al., 1986; Bittencourt et al., 1991; Day et al., 1993). Be- cause SP is colocalized in 15–25% of the A1 catecholamine neurons (Bittencourt et al., 1991), it is a candidate for chemical transmission by this pathway. The current finding of hypotension-induced NK3-R internalization is consistent with this possibility. Ligand-induced internalization of NK3-Rs is mediated by ␤-arrestin 2 in NK3-transfected human embryonic kidney 293 cells, resulting in rapid recycling to the cell membrane and resen- sitization of the cells to an NK3 ligand (Schmidlin et al., 2003). The time course of change in cytoplasmic localization of NK3-Rs in SON neurons induced by hypotension is consistent with rapid Figure 10. Quantification of nuclear localization of NK3 immunoreactivity. A, After microin- resensitization, because a significant increase in cytoplasmic NK3 jection of senktide, but not PBS and SP, the number of neurons with NK3 immunoreactivity in immunoreactivity was apparent as early as 5 min after hydral- the nucleus was significantly increased (*p ϭ 0.002 injected vs noninjected side). n ϭ 9–12 azine, but it returned to basal levels within 15 min. The latter animals per group. B, Hydralazine-induced hypotension significantly increased the number of neu- could indicate rapid recycling to the cell membrane, and rapid rons with an NK3-IR-positive nucleus at 5, 15, and 60 min compared with saline-injected normoten- ϭ ϭ ϭ receptor resensitization is consistent with the continued secretion sive animals ( p 0.02 at 5 and 15 min; p 0.05 at 60 min). n 4 or 5 animals per group. of VP observed during senktide perfusion of HNS explants; how- ever, translocation of NK3 immunoreactivity to the nucleus was also clear localization was accomplished by counting the number of observed and could contribute to the decrease in cytoplasmic NK3 SON neurons in which NK3-IR particles were confirmed to exist immunoreactivity observed after 15 min of hypotension. within the nuclear boundaries by examining the z-stack of con- The observed translocation of NK3 immunoreactivity to the focal images through each cell body. As shown in Figure 10, mi- nucleus was unexpected, because classically, GPCRs are believed croinjection of senktide, but not PBS or SP, induced nuclear to alter cellular processes by initiating signal transduction cas- localization of NK3-IR ( p ϭ 0.002; injected vs noninjected side). cades from the cell membrane. After internalization into endo- There was also a significant increase in the number of NK3- somes, most GPCRs are either recycled to the membrane result- positive nuclei at all time points evaluated in the hypotensive ing in resensitization of the cell to the ligand or transferred to animals ( p Ͻ 0.05). These observations suggest that in addition lysosomes for degradation (Kobilka, 1992; Mantyh et al., 1995); to translocation of NK3-Rs from the cell membrane into cyto- however, other GPCRs have been shown to enter the nucleus. plasmic endosomes, there is also translocation into the cell These include the angiotensin 1 (AT1) receptor (Lu et al., 1998) nucleus. as well as the closely related apelin and 2 receptors (Lee et al., 2004) and the parathyroid (Watson et Discussion al., 2000). The fact that nuclear localization of NK3 immunore- These studies demonstrate that a specific NK3-R agonist, senk- activity was increased by the manipulations that induced NK3 tide, and a physiological stimulus for VP and OT secretion, hy- internalization, e.g., senktide injection or hypotension, but not potension, induce NK3-R activation in SON as evidenced by in- by SP injection, supports the hypothesis that nuclear NK3 immu- ternalization of NK3 immunoreactivity. In addition, the in vitro noreactivity reflects ligand-activated NK3 that is translocated studies confirmed the in vivo evidence from other laboratories from the cell membrane to the nucleus. that activation of NK3-Rs by senktide stimulates VP secretion The presence of GPCR in the nucleus raises important ques- from the neural lobe (Eguchi et al., 1996). These observations tions. How do they get into the nucleus? What is their role in the suggest that release of an endogenous tachykinin in SON contrib- nucleus? These issues have been considered most extensively for utes to hypotension-induced stimulation of VP and OT release; the AT1 receptor (Lu et al., 1998). In considering the first ques- however, the endogenous ligand for the NK3-Rs in SON remains tion, the AT1 receptor was found to have a nuclear localization to be identified. sequence (NLS) in its cytoplasmic C tail (Lu et al., 1998). An NLS Internalization of NK1-R has been used extensively to study is a sequence of amino acids that exists in proteins that are found the role of SP in physiological responses (Sabino et al., 2002; in the nucleus. The consensus bipartite NLS consists of a 10–12 Howe et al. • Role of Neurokinin 3 Receptors in Supraoptic Neurons J. Neurosci., November 10, 2004 • 24(45):10103–10110 • 10109 amino acid spacer flanked by repeats of basic amino acids [lysine increase in plasma VP after intracerebroventricular injection of or arginine; K/R-K/R– 10–12 aa spacer –K/R-K/R-K/R-K/R SP but not senktide at similar concentrations (Polidori et al., (Jans and Hubner, 1996)]. Such a sequence is present in the clas- 1989; Spitznagel et al., 2001). Another possibility is that SP may sic steroid hormone receptors that concentrate in the nucleus and activate cellular processes via NK3-Rs independent of inducing function as transcription factors. It is also observed in other nu- receptor internalization. Dissociation between receptor signaling clear proteins such as the transcription factor, c-fos, and and endocytosis has been shown for morphine activation of ␮ poly(ADP-ribose) polymerase (Jans and Hubner, 1996). Exami- opiate receptors (Whistler et al., 1999). The failure of SP to in- nation of the NK3 sequence (Buell et al., 1992; Takahashi et al., duce NK3 internalization also prevented SP-induced transloca- 1992), revealed a similar sequence in the cytoplasmic C terminus tion of NK3-R to the nucleus. Thus, if there is a dissociation of the rat NK3-R in which the sequence of amino acids 428–446 between SP-induced NK3 signaling and receptor internalization, is RKKRATPRDPSFNGCSRR. In the case of the AT1 receptor, it would suggest that the potential for NK3 regulation of gene mutations in the NLS prevented nuclear translocation of the recep- expression is ligand dependent; however, if dissociation between tor (Lu et al., 1998). Similar experiments with the NK3-R are re- receptor activation and endocytosis occurs with SP activation of quired to confirm that the identified sequence functions as an NLS. NK3-Rs in SON, it would not be universal to NK3-Rs, because Nuclear translocation of NK receptors has not been reported incubation of brainstem slices with 5 ␮M SP did induce NK3-R previously despite the extensive studies on the internalization internalization (Colin et al., 2002). Additional endpoints such as and recycling of these receptors (Grady et al., 1995; Mantyh et al., calcium imaging may be useful in determining whether SP acti- 1995; McConalogue et al., 1998; Jenkinson et al., 2000; Colin et vates NK3-Rs in SON without inducing receptor internalization. al., 2002; Sabino et al., 2002; Marvizon et al., 2003). This is not unexpected for the NK1-R because it does not contain a putative References NLS (Takeda et al., 1991; Takahashi et al., 1992). 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